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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑 : F# Q+ V t) k9 s+ C
changbaihou 发表于 2013-9-3 10:38 
+ w) v J8 y5 D2 e6 G4 w多谢回复，然后呢？怎么用OCT包埋？% U3 m! q, `9 p" K8 R2 T- K. n
请问哪里可以找到protocol啊？
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9 [( a4 @1 t bPreparation of neurospheres for cryostat sectioning and immunocytochemistry:, X2 W/ {2 G0 \) t$ h, ~
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Procedures have been developed to section intact neurospheres. e8 u9 E2 M+ s
0 G( U; Z9 z3 f: v7 i1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.8 X/ [" ~# p/ t& L2 Q
2. Allow the neurospheres to settle by gravity (approximately 30 minutes. The time for the neurospheres to settle is dependent on the size of the neurospheres./ S6 n3 R v- K2 t, \
; ]" i# C9 N2 `. E& nCaution0 Q3 r) A: I) H1 u. m
Centrifugation of the viable neurospheres will alter the shape of the neurospheres.
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3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).
* w% A. X+ m; ?! {# p! z* Q6 n4. Allow the neurospheres to settle in the tube.$ b( J: X9 P5 ?' `' f. E R
5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS).
7 l7 ~9 p1 O: }6 N8 V' J6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.
- e, P* }# _( r: P2 D& i/ m# z7. Repeat the rinsing procedure at least 3 times.4 ]+ \ R7 n+ U' p) x0 `
Immunocytochemistry may be performed on intact neurospheres at this stage.
* O5 d$ G: O; K; H! b1 RTIP: Observing the removal of the supernatant at any step under the dissecting scope ensures maximum withdrawal of the fluid without disturbing the cell pellet.
) c: M/ ?3 i7 ?8 w5 J8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).
1 Y0 v/ G+ ]/ R3 e: h7 i9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.: M; J. ~* [6 g# F0 T
10. Remove the 30% sucrose and add embedding medium for frozen tissue specimens for 1 hour (Sakura Tissue-Tek O.C.T. compound, cat. no. 4583)
: ?7 h! [% W' a" b1 n& G) b11. Prepare the cryostat chuck with a layer of O.C.T.
$ M4 _% W# r" w( g K# J12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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Caution* h' d$ C9 J- x
You can lose the neurosphere pellet quickly during the transfer of the pellet from the tube to the cryostat chuck. With a Pasteur pipette or an 1000 μl Eppendorf pipettor (using the blue tips) pre-load a small amount of O.C.T. into the tip and then withdraw the neurosphere pellet into the tip, continuous with the pre-loaded O.C.T. Try not to induce any air bubbles into the O.C.T.& a( r7 L! I" W0 P3 i9 i
' I( m B# Y- h& J; N* \13. Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (Sigma cat. no. A3648) coated glass microscope slides.
3 R4 o! ~5 K+ l14. Continue with the desired immunocytochemical procedure (non-specific blocking, application of primary antibody etc.). |
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发表于 2013-9-1 07:58
